Treponema denticola (Td) is strongly implicated as a major participant in periodontal diseases, which cause tooth loss and are implicated in systemic diseases including cardiovascular disease. Td is similar to the syphilis spirochete T. pallidum, which is a major cause of morbidity and mortality world-wide. Both Td and T. pallidum have developed resistance to specific antibiotics. The long term goal of this research is to develop new drugs to treat spirochetal diseases, with Td serving as the model system. Td and other spirochetes are highly invasive bacteria due to their unique mode of motility. Spirochete periplasmic flagella (PFs) are essential for motility and virulence. The hook structure is an essential component of all bacterial PFs. The hook consists of multiple FlgE proteins. The central hypothesis is that the FlgE proteins of Td and other spirochetes is covalently cross-linked to strengthen the hook for optimal motility and virulence. Other bacteria with external flagella lack cross-links. Understanding the structure and synthesis of FlgE cross-links could lead to development of novel drugs that inhibit cross-linking to treat periodontal disease, syphili and other spirochetal diseases. The three aims below address this hypothesis. Specific aim 1. We hypothesize that the Td FlgE flagellar hook proteins are covalently cross- linked. To test this hypothesis, we will determine the precise chemical structure of the cross-link using hook proteins isolated from Td and cross-linked FlgE proteins formed in vitro. The approach utilizes mass spectrometry and how cross-linking is deciphered in other systems. Specific aim 2. We hypothesize that site-directed mutations in Td FlgE will result in the inability of the hook protei to be cross-linked both in vitro and in vivo. The approach incorporates in vitro mutagenesis of rflgE, and introducing select mutations into Td cells and analyzing their phenotypes. Cells bearing such mutations are hypothesized to have altered motility. Specific aim 3. We hypothesize that cross-linking FlgE is essential for Td to cause disease. To test this hypothesis, we will analyze mutants defective in cross-linking and make comparisons to the wild-type using two established virulence assays. The results obtained should allow us to determine the role of FlgE cross-linking in Td pathogenesis.